The final stage of sperm development, spermiogenesis, is essential for formation of viable spermatozoa;however, the molecular mechanisms underlying this process are largely unknown. During sperm maturation, an extraordinary morphological change in the gamete occurs converting the round spermatid into a streamlined motile sperm. Essential to this transformation is the manchette, a unique skirt-like structure composed of numerous microtubules that surrounds the spermatid nucleus, constricts in diameter while it translocates along the nuclear membrane. The manchette may function by directing mechanical force on the nucleus necessary for shape change, by providing tracks for transport of cytoplasmic contents, or by supplying a platform for the localization of signaling molecules. We have recently obtained evidence supporting this latter function for the manchette. We have identified a new testis-specific protein, named testis leucine rich repeat (TLRR), which is linked to the spermatid manchette and is homologous to a regulatory subunit of protein phosphatase-1 (PP1). PP1 activity is essential for successful spermiogenesis. Our preliminary studies indicate that TLRR is localized near the spermatid nucleus during the time of its greatest morphological change and that TLRR interacts specifically with PP1 isoforms in the testis. The central hypothesis to be tested in this application is that TLRR is a scaffolding protein that functions to localize signaling molecules, including PP1, near the nucleus of male germ cells in order to regulate specific proteins necessary for transformation. This hypothesis will be addressed through 2 specific aims: (1) to examine the molecular interaction of TLRR and PP1, and (2) to determine whether this interaction affects the localization and/or activity of PP1. In the first aim, these two proteins will be colocalized in developing germ cells using both electron and confocal microscopy. Coimmunoprecipitation experiments will determine whether the TLRR-PP1 interaction is isoform specific and whether binding is restricted to specific cell types in the testis. The importance of distinct protein domains and sequences for interaction will be assessed by molecular genetic approaches. In the second aim, experiments will establish whether the localization and/or enzymatic activity of PP1 is affected by its binding to TLRR and whether the phosphorylation state of either partner influences their interaction. The experiments described in this proposal will provide an important link between a scaffolding protein located near the transforming spermatid nucleus and a central signaling molecule, PP1, and lay the foundation for an R01 application to more fully investigate the role of regulatory and structural proteins in spermatid transformation. This research will examine the importance of a testis-specific regulatory protein in sperm formation and therefore will advance possible remedies for male infertility as well as new strategies for contraceptive design. PUBLIC HEALTH RELEVANCE: Mammalian sperm result from a series of carefully orchestrated cellular events that transform a precursor germ cell into a highly specialized haploid gamete that can effectively deliver genomic information to the oocyte at fertilization. Failure at any step in sperm formation can result in dysfunctional sperm with abnormal shape, lack of motility, absence of necessary subcellular structures, or presence of chromosomal abnormalities. This research will examine the importance of a testis-specific regulatory protein in spermatogenesis and will provide insight into possible remedies for male infertility as well as possible strategies for contraceptive design.